PROJECT SUMMARY Intra-tumoral heterogeneity in human cancers is associated with poor treatment response and prognosis. Head and neck squamous cell carcinoma (HNSCC) is a strikingly heterogeneous tumor, with over 550,000 new cases per year. Unfortunately, this heterogeneity represents a major challenge in targeted therapy and treatment responses, with detailed maps of intra-tumoral heterogeneity just starting to be defined. During my Ph.D. training with Dr. Azad Bonni, I studied normal processes of brain development using sophisticated biochemistry techniques1,2,3,4, which inspired me to understand the signaling pathways that drive specific biological programs. I applied these skills to studies of glioma, ultimately, defining a dual role for the transcription factor STAT3 as a tumor suppressor and an oncogene5,6,7. For my post-doctoral training, I joined the laboratory of Dr. Bradley Bernstein, an expert in transcription, epigenetics, and innovative technology, to combine my interests in tumor heterogeneity with my clinical interests in head and neck surgical oncology. Using single cell RNA-seq, we defined the first atlas of HNSCC, identifying a partial epithelial-to-mesenchymal (p-EMT) program associated with lymph node metastasis, lymphovascular invasion, and extracapsular extension8. We also refined tumor subtyping from bulk RNA-seq approaches such as the Cancer Genome Atlas by leveraging our single cell profiles. The objectives of this proposal are to (1) determine expression heterogeneity within 2D and 3D head and neck cancer models and identify suitable models of p-EMT, (2) investigate the regulatory influence of cancer- associated fibroblasts and their ligands on malignant cancer cell states, including the underlying transcriptional and epigenetic mechanisms, and (3) define the signaling pathways downstream of p-EMT and discern their functional importance in tumor behaviors. Dr. Tim Ley and Dr. Greg Longmore are exceptional mentors with an extensive history of trainees in major academic positions. Dr. Ley is an internationally renowned leader in oncology, genomics, and tumor biology, with appointments including Associate Director of the Siteman Cancer and the McDonnell Genome Institute at Washington University St. Louis (WUSTL), National Cancer Advisory Board Member, and former Chair of Board of Scientific Counselors for National Institute Genome Research Institute. Similarly, Dr. Greg Longmore is a world expert in molecular oncology, EMT, and cell biology, with appointments as Co-Director of Molecular Oncology and Director of the Integrating Communications within the Cancer Environment Institute, WUSTL. An exceptional Research Advisory Committee (RAC) will provide additional guidance, including Dr. Ting Wang, Dr. Jose Zevallos, and Dr. Rob Mitra. The K08 award will provide me with the ideal opportunity to succeed in my career goals. The detailed training plan includes a RAC and development of technical/leadership skills that will be invaluable for a transition to independence. Research will be carried out at WUSTL, a prestigious research institute that fosters collaboration, emphasizes intellectual exchange, and boasts unparalleled facilities. 1. Kim AH, Puram SV, Bilimoria PM et al. Cell 136(2): 322-336 (2009). 2. Puram SV, Kim AH, Ikeuchi Y, et al. Nature Neuroscience 14(8): 973-983 (2011). 3. Puram SV, Riccio A, Koirala S et al. Genes and Development 25(24): 2659-2673 (2011). 4. Puram SV*, Kim AH*, Park HY et al., Cell Reports 4(1): 19-30 (2013). 5. de la Iglesia N, Konopka G, Puram SV et al., Genes and Development 22(4): 449-462 (2008). 6. Puram SV, Yeung CM, Asl-Jahani A, et al. Journal of Neuroscience 32(23): 7806-7818 (2012). 7. Jahani-Asl A, Yin H, Soleimani VD et al., Nature Neuroscience 19(6): 798-806 (2016) 8. Puram SV*, Tirosh I*, Parikh A* et al., Cell 171(7): 1611-1624 (2017)